Figure 5.

Acetylation and polyglutamylation of centrioles and cilia. Figure illustrates centrioles and basal bodies with cilia from cultured cells. Cells were expanded 4.2-fold (as detailed in Ref. [237]) and imaged by structured illumination microscopy (SIM, A–D) or conventional widefield microscope using 60× lens (E). Centrioles and cilia were co-immunolabeled with antibodies recognizing acetylated tubulin (Sigma; T7451) and centriole ‘cap’ protein Cep290 (Abcam; ab84870) or polyglutamylated tubulin (using an antibody which recognizes a chain of 4 or more glutamates, Adipogen (rabbit, AG-25B-0030-C050)). (A) Duplicated older mother centriole from mIMCD3 (mouse inner medullary collecting duct) cell associated with a cilium. Both centrioles (blue arrows) and the ciliary axoneme, (red arrows) are acetylated. Mother centriole and ciliary axoneme are also polyglutamylated. (B) Duplicated mother centrioles from HeLa cells associated with short procentrioles. Cep290 ‘caps’ distal ends of centrioles (blue arrows). Note that very short procentrioles are already acetylated. (C) G1 centrioles from RPE-1 cells. Mother centrioles are acetylated along the entire MT length but polyglutamylation signal is not present on distal ends. Daughter centrioles still lack or have low levels of polyglutamylation in G1 (yellow arrows). (D) Examples of centrioles from mIMCD3 cells. Mother centrioles are acetylated and polyglutamylated. Polyglutamylation signal associated with procentrioles is low (yellow arrows). (E) Examples of two duplicated mother centrioles from a HeLa cell in S/G2 phase. One (older) mother centriole is more polyglutamylated than the younger mother centriole. Scale bars: 1000 nm.